(Th1) and Th17 Self‐Reactive Immune Respon

BJU Int 2020; 126: 379–387 doi:10.1111/bju.15117

Original Article

Patients with chronic prostatitis/chronic pelvic pain syndrome show T helper type 1 (Th1) and Th17 self-reactive immune responses speciﬁcto prostate and seminal antigens and diminished semen quality Ruben D. Motrich1 , Marıa L. Breser1, Rosa I. Molina2, Andrea Tissera2, Jose J. Olmedo3 and Virginia E. Rivero1 1Centro de Investigaciones en Bioquımica Clınica e Inmunologıa (CIBICI-CONICET), Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba, Cordoba, Argentina, 2Laboratorio de Andrologıa y Reproduccion (LAR), Cordoba, Argentina, and 3Fundacion Urologica Cordoba para la Docencia e Investigacion Medica (FUCDIM), Cordoba, Argentina

Objectives To assess the presence of self-reactive immune responses to seminal and prostate antigens (PAg), biomarkers of inflammation of the male genital tract, and semen quality parameters in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). Patients, Subjects and Methods Peripheral blood and semen samples were collected from patients with CP/CPPS and age-matched healthy control volunteers. We analysed the lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) to different seminal plasma (SP)-derived and purified PAg, serum autoantibodies specific to PAg, leucocyte subpopulations, and inflammatory cytokines in semen, sperm apoptosis/necrosis, and semen quality parameters. Results Significantly greater PBMC proliferative responses specific to PAg, with elevated secretion of interferon (IFN)c and interleukin (IL)-17, were detected in the patients with CP/CPPS vs the controls. Moreover, the patients with CP/CPPS had significantly greater serum immunoglobulin G immune reactivity to SP proteins, such as prostate-specific antigen and prostatic acid phosphatase, than the controls. Inflammation of the male genital tract was exemplified by high levels of IFNc, IL-17, IL-1b and IL-8, as well as higher counts of leukocytes, mainly CD4 T lymphocytes and macrophages, in the semen. In addition, this local inflammation was associated with an overall diminished semen quality, i.e., reduced sperm concentration, motility and viability; and higher levels of sperm apoptosis/necrosis in patients with CP/CPPS vs controls. Conclusion Patients with CP/CPPS show T helper type 1 (Th1) and Th17 immune responses specific to PAg associated with chronic inflammation of the male genital tract and reduced semen quality. These immune responses may underlie the induction and development of chronic pelvic pain and inflammation of the male genital tract, which in turn could alter normal prostate functioning and impair semen quality. Keywords prostatitis, inflammation, autoimmunity, MaleInfertility, semen analysis

region, painful DRE, negative Meares–Stamey test, no other Introduction lower urinary tract pathology, and for ≥3 of the preceding Prostatitis is a major medical problem, being the most 6 months a CP/CPPS history >1 year, and a NIH Chronic common urological diagnosis in men aged <50 years and the Prostatitis Symptoms Index (NIH-CPSI) total score >15. third most common in older men [1]. Prostatitis is currently Control subjects had no history of any genitourinary classified into four categories by the National Institutes of symptoms, instrumentation or surgery. Exclusion criteria Health (NIH): acute bacterial prostatitis, chronic bacterial included specific disease-associated pelvic pain/discomfort prostatitis, chronic non-bacterial prostatitis/chronic pelvic caused by non-CP/CPPS diseases (e.g. acute prostatitis, pain syndrome (CP/CPPS), and asymptomatic inflammatory bacterial prostatitis, BPH, prostate cancer, urogenital infection), prostatitis [2]. CP/CPPS accounts for >90% of cases of serious or acute disease of the heart, liver, kidney or blood, prostatitis and affects 8–14% of men of all ethnic origins vasectomy, toxic/pollutant exposure, any drug, alcohol or [3,4]. CP/CPPS has a considerable negative impact on quality marijuana consumption, antibiotic, steroids or NSAID of life, similar to myocardial infarction, angina, or Crohn’s treatment during the preceding 12 weeks. Patients and controls disease [4]. It is a poorly understood clinical syndrome were prospectively included in the study and they donated characterised by local signs and symptoms of chronic blood and semen samples. This study was carried out in inflammation and genitourinary pain (defined as lasting ≥3of accordance with the Declaration of Helsinki and the the preceding 6 months) in the absence of identifiable Argentinean legislation for protection of personal data (Law urogenital infections [1]. As its aetiology remains obscure, 25326). The experimental protocol was approved by the most therapies are empiric and ineffective [5]. CP/CPPS Institutional Ethics Committee from the Hospital Privado de appears to encompass similar clinical phenotypes resulting Cordoba (Ref. #HP-4-132). All participants provided a signed from a wide array of heterogeneous conditions. It has been written informed consent prior to enrolment. suggested that dysregulated inflammation resulting from autoimmunity against the prostate might be involved in its Lymphoproliferation Assays onset and/or progression [6]. Indeed, no infectious cause has been successfully identified to date and treatments with Peripheral blood samples were obtained and collected in fi corticosteroids or immunosuppressive drugs, rather than with sterile heparinised tubes. PBMC were puri ed by density antibiotics, have been shown to be effective [5,7–9]. gradients (Ficoll-Paque PLUS, GE Healthcare Life Sciences, fi Moreover, the prostate-specific protein transglutaminase-4 Pittsburgh, PA, USA) and lymphoproliferation assays speci c (TGM4) has been identified as a target of the autoimmune to SP (SP0, human prostate homogenate (PHg), PSA and response in autoimmune polyendocrine syndrome type 1 prostatic acid phosphatase (PAP) were performed as (APS1), which presents with clinical and histopathological previously described [15]. In brief, PBMC were divided as prostatitis and subfertility in males [10]. Regarding the latter, responder or antigen-presenting cells (APC). APC were some recent evidence suggested that CP may have loaded with different PAg sources: dilutions (1:25) of pooled µ µ andrological implications, as it could have deleterious effects SP from normal donors, PHg (100 g/mL), PSA (10 g/mL, µ on semen quality compromising male fertility potential [11– Sigma-Aldrich, St. Louis, MO, USA), PAP (10 g/mL, Sigma- 13]. Nonetheless, that issue remains controversial highlighting Aldrich), or medium alone, and then irradiated at 3000 cGy. 9 5 the need for additional and compelling data [14]. Pulsed APC were added to plates containing 1.5 10 responder PBMC in quadruplicate and incubated for 96 h at ° µ In an effort to gain further understanding of the 37 C/5% CO2. Concanavalin A (5 g/mL, Sigma-Aldrich) pathophysiology underlying CP/CPPS, in the present study, we was used as a positive control of proliferation. Supernatants analysed the presence of self-reactive immune responses to were collected and frozen at À80 °C for cytokine seminal plasma (SP)-derived and prostate antigens (PAg), quantification. DNA synthesis was measured by adding inflammatory cytokines and leucocyte subpopulations in the 37000 Bq of [methyl-3H] thymidine (Perkin Elmer, Waltham, semen, and sperm quality parameters in patients with CP/CPPS. MA, USA) in fresh medium per well 18 h before harvesting cells onto glass fibre filters. Labelled material was counted and results expressed as a Proliferation Index (PI) calculated Patients, Subjects and Methods from counts/min (cpm) incorporated in antigen-pulsed Patients and Subjects cultures/cpm incorporated in cultures with medium. A PI of ≥ CP/CPPS was defined as pain or discomfort in the perineum 2.00 was considered positive. and the suprapubic region with LUTS or sexual/ejaculatory dysfunction, without infection [2]. Diagnosis was based on a PAg-specific Antibodies in Serum detailed history, physical examination, ultrasonography, and Immunoglobulin G (IgG) antibodies specific to SP, PSA or – laboratory evaluations. Inclusion criteria were men aged 18 PAP were assayed by conventional ELISA as previously 55 years with history of pain or discomfort in the prostate

described [15]. Serum reactivity was expressed as optical Sperm apoptosis/necrosis density at 450 nm. Sperm apoptosis/necrosis was assessed immediately after Semen analysis semen liquefaction by annexin V/propidium iodide staining and analysed by flow cytometry as previously described [18]. Semen samples were obtained by masturbation after 4–7 days of sexual abstinence and semen analysis performed according Statistics to the WHO Semen Analysis Manual [16]. Semen analysis was performed at least twice in each patient and control. The Statistical analysis was performed using the Mann–Whitney sperm concentration and motility were evaluated soon after test. A P < 0.05 was considered statistically significant. liquefaction in a Makler chamber (Sefi-Medical Instrument, Compromise power analyses were calculated using G*Power3 Haifa, Israel) on a phase-contrast microscope. Sperm viability data analysis software. Considering q: 0.5 and a: 0.05, the and morphology were analysed using eosin Y (Sigma-Aldrich) statistical power of the study (1–b) was 0.68. staining and the Papanicolaou technique, respectively. The concentration of round cells was evaluated using the Makler chamber and peroxidase-positive cells were quantified among Results round cells using a cytochemical assay [17]. Semen citrate Detection of PAg-specific T helper type 1 (Th1) and and fructose concentrations were measured as biomarkers of Th17 immune responses in patients with CP/CPPS prostate and seminal vesicles function, respectively, by To search for the presence of immune reactivity against PAg colorimetric assays. Anti-sperm IgG antibodies were tested in patients with CP/CPPS, we analysed peripheral blood using the direct SpermMAR test (FertiPro, N.V., Beernem, immune responses in the patients and controls. A total of 15 Belgium). Routine sperm parameters were assessed in at least patients [mean (SD, range) age 40.3 (7.4, 30–55) years] and 200 spermatozoa/sample by two operators, rendering a total 10 controls [mean (SD, range) age: 37.7 (6.4, 24–45) years] of 400 scored spermatozoa. were included. As shown in Figure 1A, significantly greater proliferative responses were detected when PBMC from Semen Leucocyte Analysis by Flow Cytometry patients with CP/CPPS were in vitro stimulated with different sources of PAg (SP or PHg) or purified PAg (PSA and PAP) Leucocyte subpopulation analysis was performed in total vs controls. Indeed, 53.3% (eight out of 15) of the patients semen single-cell suspensions obtained after washing semen had positive lymphoproliferative responses to one or more of aliquots with 10% fetal bovine serum, 2 mM EDTA, and the PAg assayed. Moreover, when assaying PAg-specific 50 mM 2-mercaptoethanol supplemented Roswell Park cytokine secretion in culture supernatants, higher levels of Memorial Institute (RPMI)-1640 medium. Cell suspensions specific IFNc and IL-17 secretion to one or more of the PAg fl were stained with uorescent-labelled antibodies to cluster assayed were detected in cell cultures from the patients with of differentiation (CD)45, CD3, CD4 and CD19 CP/CPPS compared with the controls (Figure 1B). (BioLegend, San Diego, CA, USA), and to CD14 Conversely, lower levels of PSA- and PAP-specific IL-10 (eBioscience, San Diego, CA, USA). Cells were acquired secretion were observed (Figure 1B). On the other hand, fl fl and analysed by uorescence-activated cell sorting/ ow patients with CP/CPPS had higher serum levels of SP-, PSA-, cytometry (FACS) on FACSCanto II cytometer and PAP-specific IgG autoantibodies than the controls (BD Biosciences). A total of 100 000 cells/sample were (Figure 1C). Also, significantly higher levels of serum PSA analysed. were found in patients with CP/CPPS than in the controls, suggesting a state of inflammation of the prostate Cytokine and PSA Quantification (Figure 1D). These results indicate that patients with CP/ Interferon (IFN)c, interleukin (IL)-10, IL-8, IL-17A and IL-1b CPPS have Th1 and Th17 self-reactive immune responses fi concentrations in SP or culture supernatants were analysed and also autoantibodies speci ctoPAg. using ELISA specific kits. IFNc, IL-10 and IL-8 were fi TM c respectively quanti ed by the BD OptEIA Human IFN ,IL- Patients With CP/CPPS Show Local Inflammation in 8 and IL-10 ELISA sets (BD Biosciences, cat. # 555142, the Male Genital Tract 5126542 and 555157). IL-17A and IL-1b were respectively assayed by the Human IL-17A ELISA Ready-SET-Go and As shown in Figure 2A, flow cytometry analysis of semen Human IL-1b ELISA Ready-SET-Go kits (eBioscience, cat. # samples revealed that the patients with CP/CPPS had 88-7010 and 88-7176). Serum PSA concentrations were significantly more leucocyte s in their semen when compared determined using chemiluminescence immunometric assay with the controls. When analysing leucocyte subpopulations, kits (Immulite, Malvern, PA, USA). macrophages and CD4+ T helper cells were the most

Fig. 1 Self-reactive Th1 and Th17 immune responses speciﬁc to PAg in patients with CP/CPPS. (A) Lymphoproliferative responses of PBMC from patients with CP/CPPS and age-matched control subjects to different SP-derived and PAg: dilutions (1:25) of SP obtained from pooled donor samples having a normal semen analysis, PHg, PSA, PAP, or medium alone. Lymphoproliferative responses were expressed as PI calculated from counts/min (cpm) incorporated in antigen-pulsed cultures/cpm incorporated in cultures with medium. (B) INFc, IL-17 and -10 secretion of PBMC from patients with CP/ CPPS and age-matched control subjects cultured with the same PAg sources assayed by sandwich ELISA. (C) Serum levels of IgG antibodies speciﬁcto SP, PSA and PAP in patients with CP/CPPS and age-matched control subjects examined by indirect ELISA in 1:50 serum dilutions. Serum reactivity was expressed as optical density (OD) at 450 nm. (D) Serum levels of PSA in patients with CP/CPPS and age-matched control subjects assayed by chemiluminescence immunoassays and results expressed as ng/mL. Experiments were performed at least in triplicate and repeated twice with similar results. Data are shown as mean Æ SD; patients with CP/CPPS, n = 15; age-matched control subjects, n = 10. Mann–Whitney test; *P < 0.05, **P < 0.01 and ***P < 0.005.

A C D 5 ** * 20 *** * ** ** *** 4 0.8 15

3 0.6 10 2 0.4 OD (450 nm) PSA (ng/mL) 5 Proliferation index 1 0.2

0 0.0 0 SP SP SP SP PHg PSA PAP PHg PSA PAP PSA PAP PSA PAP Controls Patients Controls Patients ControlsPatients

B * ** 800 * 400 * 400 * 600

400 300 300 400 (pg/mL) (pg/mL)

(pg/mL) 200 200 γ −17 −10 IFN 200 IFN 100 IFN 100

0 0 0 SP SP SP SP SP SP Basal PHgPSAPAP Basal PHgPSAPAP Basal PHgPSAPAP Basal PHgPSAPAP Basal PHgPSAPAP Basal PHgPSAPAP Controls Patients Controls Patients Controls Patients prominent cell subsets. Moreover, significantly higher levels of alterations in semen volume, pH values, fructose levels and inflammatory cytokines, such as IFNc, IL-17, IL-1b and IL-8, peroxidase-positive cell concentrations were found in the were detected in the SP from patients with CP/CPPS patients with CP/CPPS compared with the controls. compared with the controls (Figure 2B). These results Moreover, no presence of anti-sperm antibodies was observed indicate a state of chronic inflammation in the male genital in either the patients or controls (Table 1). Conversely, tract of patients with CP/CPPS, probably due to PAg-reactive significantly lower seminal citrate levels were detected in the immune responses. patients than in the controls (Table 1). These data indicate that the inflammatory milieu in the urogenital tract in patients with CP/CPPS might impair prostate physiology. Patients With CP/CPPS Have Decreased Semen Sperm quality parameters analysis showed significantly lower Quality sperm concentration, motility and viability, and higher counts To analyse if local male genital tract inflammation was of round cells in the patients with CP/CPPS vs controls associated with diminished semen quality, we performed (Table 1). Indeed, when sperm apoptosis/necrosis was semen analysis in the patients and controls. No significant assessed, significantly lower levels of live spermatozoa and

Fig. 2 Evidence of local inﬂammation in the male genital tract of patients with CP/CPPS. (A) Frequencies of leucocyte and different leucocyte cell subsets in semen samples from patients with CP/CPPS and age-matched control subjects. Total leucocytes (CD45+), T lymphocytes (CD3+), helper T lymphocytes (CD3+ CD4+), cytotoxic T lymphocytes (CD3+ CD4–), macrophages (CD14+) and B lymphocytes (CD19+) were assayed in semen samples by ﬂow cytometry. Analyses were performed in gates on the total leucocyte cell population (CD45+) using forward side scatter (FSC) vs CD45 dot plots. A total of 100 000 cells/events per specimen were analysed. Results were expressed as % of cells. (B) INFc, IL-17, IL-1b and IL-8 levels in SP samples from patients with CP/CPPS and age-matched control subjects assayed by sandwich ELISA. Experiments were performed in triplicate and repeated three- times with similar results. Data are shown as mean Æ SD; patients with CP/CPPS, n = 15; age-matched control subjects, n = 10. Mann–Whitney test; *P < 0.05 and **P < 0.01.

** * A * 250K ** 45 * 200K 40 150K

100K 35

50K 30 0 25 –103 0 103 104 105 20 FSC

250K Cells (%) 15 200K 10 150K 5 100K 0 50K + ) + )

0 B cells B cells –103 0 103 104 105 Leucocytes CD8 T cells Leucocytes CD8 T cells T cellsCD4+ (CD3 T cellsMacrophages T cellsCD4+ (CD3 T cellsMacrophages CD45 Controls Patients

B **** ** 30 30 30 1200

25 25 25 1000

20 20 20 800

15 15 (pg/mL) 15 600 (pg/mL) β γ

IFN 10 IL-17 (pg/mL) 10 IL-1 10 IL-18 (pg/mL) 400

5 5 5 200

0 0 0 0

Controls Pateints Controls Pateints Controls Pateints Controls Pateints

higher levels of early apoptotic and late apoptotic/necrotic spermatozoa were found in the patients vs controls Discussion (Figure 3). Although CP/CPPS is the most common type of prostatitis and genitourinary problem in adult males aged <50 years, it Overall, these results indicate that patients with CP/CPPS is still an enigmatic disease mostly due to its uncertain show Th1- and Th17-associated self-immune responses aetiology. That makes its diagnosis of exclusion, inadequate specific to PAg and higher levels of inflammatory cytokines and cumbersome. Moreover, therapies are empirical and and leucocytes in their semen. Thus, the patients had local results ineffective for most patients [5]. Over the past decade, inflammation with lower levels of biomarkers of prostate chronic inflammation has been explored as underlying physiology and reduced semen quality.

Table 1 Semen analysis in patients with CP/CPPS to bladder distention, and sensitised bladder nerve afferents, indicating cross-organ sensitisation and explaining the urinary Variable, mean (SD) Controls Patients P symptoms features of CP/CPPS. Although cryptic or difficult- Demographics to-culture micro-organisms have been suggested as putative Age, years 37.7 (6.4) 40.3 (7.4) 0.540 causative factors, studies have systematically failed to identify Abstinence, days 4.6 (3.2) 5.4 (2.4) 0.411 Semen analysis infectious agents as causative of this syndrome [22]. Volume, mL 3.2 (1.2) 4.1 (2.0) 0.438 Moreover, most patients’ symptoms are refractory to pH 7.7 (0.1) 7.6 (0.2) 0.302 antibiotic monotherapy [9]. Nonetheless, if not pathogenic, Concentration, 9106/mL 136.3 (84.6) 57.0 (58.0) 0.040 Total motility, % 65.2 (12.2) 43.1 (19.4) 0.043 infections could be the initiating factor of dysregulated Normal morphology, % 10.3 (6.4) 7.1 (3.5) 0.303 inflammation in the form of autoimmunity [23]. An Viability, % 89.5 (5.6) 82.1 (5.9) 0.034 autoimmune basis for CP/CPPS is a prominent theory based Round cells, 9106/mL 0.48 (0.28) 1.58 (0.91) 0.012 Peroxidase (+) cells, 9106/mL 0.19 (0.23) 0.81 (1.16) 0.386 on evidence obtained from studies in patients and animal Citrate, mg/dL 402.9 (54.1) 319.4 (98.1) 0.026 models [6,24]. Such animal models mirror most features of Fructose, mg/dL 349.9 (109.5) 334.2 (134.3) 0.971 human CP/CPPS and have shown that prostate inflammation Anti-sperm antibodies (ASA)* fi IgG, % sperm agglutination 0.67 (0.81) 1.64 (5.10) 0.260 induction is mediated by T-cell responses speci c to PAg. In addition, they have shown that prostate inflammation induces Mann–Whitney test; bolded values indicate statistical significance (P < 0.05). *Assayed by direct SpermMAR test. chronic pelvic pain development and has deleterious effects on semen quality compromising male fertility [6,25]. However, compelling available data from studies in patients pathogenic mechanism in CP/CPPS [6]. Prostate biopsies are limited [6,25,26]. In the present study, we provide novel revealed chronic inflammation in a considerable proportion evidence indicating that Th1 and Th17 self-reactive immune of patients and histological prostatitis was associated with a responses specific to PAg are detected in a considerable shorter time to symptom progression [19,20]. Schwartz et al. proportion of patients with CP/CPPS, which may be [21] reported that prostate inflammation significantly responsible for the observed inflammation of the male genital increased urinary voiding frequency, induced hypersensitivity tract. Significantly increased lymphoproliferative responses

Fig. 3 Patients with CP/CPPS show increased levels of sperm apoptosis/necrosis. Sperm apoptosis/necrosis was evaluated by annexin V (AV)/ propidium iodide (PI) staining and FACS. Frequencies of live (AVÀ,PIÀ), early apoptotic (AV+/PIÀ), late apoptotic/early necrotic (AV+/PI+) and late necrotic (AVÀ/PI+) spermatozoa in semen from patients with CP/CPPS and age-matched control subjects. Left, representative dot plots showing the different labelling patterns in the bivariate PI-AV analysis that identiﬁed the different cell populations designated as viable, early apoptotic cells, late apoptotic cells/early necrotic, and necrotic cells. Results are expressed as % of cells/100 000 events. Experiments were performed at least in triplicate and repeated three-times with similar results. Data are shown as mean Æ SD; patients with CP/CPPS, n = 15; age-matched control subjects, n = 10. Mann–Whitney test; *P < 0.05 and ***P < 0.005.

100 *** *** Patients * 80 27,0 17,8 104

60 103 0 40 52,3 2,9 3 4 Sperum cells (%) 0 10 10 Controls 20 12,1 1,9 Annexin V 104

0 103 0 78,2 7,7 Live (A–/IP–) Live (A–/IP–) 0 103 104 Necrotic (A–/I+) Necrotic (A–/I+) Propidium lodide Early Apoptosis (A+/I–) Early Apoptosis (A+/I–)

Late Apopt/Early NecControls (A+/I+) Late Apopt/Early Nec Patients (A+/I+)

were seen in patients when their PBMC were in vitro mediators have been proposed to trigger pain development in stimulated with different SP-derived or purified PAg. CP/CPPS [6,25]. In that regard, Th1 cells driving prostate Moreover, increased levels of PAg-specific IFNc and/or IL-17 inflammation have been shown to mediate prostate secretion were associated with these responses indicating a inflammation induction and chronic pelvic pain development Th1/Th17 mixed immune phenotype. In addition, these PAg- in an animal model of CP/CPPS [18]. In addition, Roman reactive immune responses were concomitant with the et al. [34] recently showed that prostate inflammation induces presence of local inflammation of the urogenital tract neuropathic changes and chronic pelvic pain through the evidenced by more macrophages/monocytes and CD4+ T non-selective cation channel transient receptor potential lymphocytes, high levels of IFNc, IL-17, and other pro- vanilloid 1 (TRPV1). Remarkably, IL-17 has also been inflammatory cytokines, such as IL-1b and IL-8, in the postulated to play a key role in the induction of chronic semen. Moreover, these patients had low seminal levels of pelvic pain in another animal model of CP/CPPS [35]. Our citric acid, indicative of prostate dysfunction, and alterations present results revealed the presence of Th1 and Th17 in sperm quality such as low sperm concentration, motility, immune responses specific to PAg and increased SP levels of and viability, and higher levels of sperm apoptosis/necrosis. IFNc and IL-17 in patients with CP/CPPS. Altogether, this evidence further supports the notion that IFNc and IL-17 Our present results are in agreement with the scarce reported may be involved in the onset and progression of data [27,28]. Indeed, Kouiavskaia et al. [29] showed that inflammation and chronic pelvic pain development in CP/ patients with CP/CPPS have increased frequencies of CD4+ T CPPS. cells specific to PAP. Moreover, it has been recently reported that 30% of patients with IgG4-related disease have The prostate is the major male accessory gland and symptoms of CP/CPPS and histopathological and secretes several factors that exert crucial functions for immunohistochemical evidence of prostatitis, which improve human reproduction [25,36]. Therefore, it is reasonable to after treatment with corticosteroids or immunosuppressive hypothesise that inflammation of the prostate could alter drugs [7]. Noteworthy, these patients typically develop male fertility potential [36]. In fact, prostatitis has been autoimmune pancreatitis [7]. Also, Lu et al. [30] reported postulated as a potential risk factor for male infertility that a significant proportion of patients with CP/CPPS have [11,25]. However, the impact of prostatitis on semen autoantibodies against different immunodominant prostate quality remains controversial, as conflicting results have proteins, which were able to induce autoimmune prostatitis in been reported [36]. Although some reports have shown a mice upon immunisation. Besides, and as mentioned above, negative impact of CP/PPS on sperm quality [37–39], other most patients with APS1 present elevated serum levels of studies did not reveal any difference in sperm quality autoantibodies to TGM4, indicating that prostate parameters between patients with CP/CPPS and controls autoimmunity is a feature of APS1 that could also be [40,41]. Our present results showed that patients with CP/ involved in male subfertility [10]. Chronic inflammation in CPPS had increased levels of macrophages, T lymphocytes the form of autoimmunity develops when there is continuous and inflammatory cytokines in their semen, and alterations and uncontrolled induction and activation of the immune in semen quality parameters. Flow cytometry analysis response to self-antigens, which then causes persistent injury revealed increased levels of leucocytes, mostly macrophages and tissue damage. As the inflammatory process is not and T lymphocytes, in the semen from patients with CP/ interrupted, the production of inflammatory mediators such CPPS. However, cytochemical analysis of those semen as cytokines, chemokines and reactive oxygen species persists. samples revealed no increases in peroxidase-positive cells. Autoimmune inflammation is sustained due to persistent These results highlight the need to properly interpret expression of self-antigens and by the lack of efficient control negative results when assaying leucocytes in semen by the by regulatory immune cell populations (e.g. regulatory T peroxidase assay, as it mainly identifies neutrophils but no cells), which usually bias the immune response to a other leucocyte subsets. In addition, patients with CP/CPPS destructive Th1/Th17 phenotype. In particular, Th1 cells had sperm quality alterations such as lower sperm secrete IFNc, a cytokine that causes activation of concentration, motility and viability associated with elevated macrophages, which have a dominant role in chronic levels of IFNc, IL-17, IL-1b and IL-8, and more sperm inflammation, as they contribute by secreting cytokines and apoptosis/necrosis. Cytokines such as tumour necrosis factor growth factors, such as IL-1b, IL-8, IL-23 and IL-12, further a, IL-1b, IL-6, and IL-8 have already been reported to be enhancing inflammation by activating other cells, in elevated in patients with CP/CPPS [42–44]. Moreover, IL-6 particular T cells [31]. In fact, Th1 and Th17 cells are known and IL-8 have been proposed as biomarkers of urogenital to drive the pathogenesis of many autoimmune diseases [32]. inflammation [45–47]. Nonetheless, and to the best of our In addition, IL-17 has been shown to be involved in knowledge, we report for the first time elevated levels of inflammatory and neuropathic pain induction [33]. IL-17 and IFNc in urogenital secretions from patients with Neurogenic processes, immune injury and inflammatory CP/CPPS [42].

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